RhoA is a Key Facilitator of Airway Inflammation and Asthma

Introduction

Airway inflammation and intermittent narrowing of airways are key characteristics of the chronic lung condition known as asthma, which, due to an increasing prevalence, is now estimated to affect 300 million people worldwide^{(reviewed in 1)}. Standard therapeutic interventions for asthma include corticosteroids for managing chronic disease changes and β2‐adrenoreceptor agonists for acute asthma attacks. While they provide many benefits, these interventions do not correct all pathological changes to the remodeling airways associated with asthma. One of the key cellular targets in asthma is airway smooth muscle cells (ASMCs), whose contraction is controlled via Ca2+-dependent and Ca2+-sensitization mechanisms^{(reviewed in 2)}.As discussed in the review by Chiba et al., one of the primary mechanisms regulating Ca2+-sensitization in ASMC contraction is the small GTPase, RhoA.Recently, several studies have expanded our understanding of RhoA’s role in regulating asthma through airway hyperresponsiveness (AHR) and ASMC contraction, as well as through other asthma pathological features such as β‐adrenergic desensitization and airway inflammation^{(reviewed in [3])}. This newsletter will discuss recent findings about the role of RhoA in the pathophysiology of asthma.

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RhoA regulates ASMC hyperresponsiveness, contraction, and remodeling in asthma

The small GTPase RhoA functions as a molecular switch oscillating between an active state when bound to GTP and an inactive state when bound to GDP.Canonical RhoA signaling involves the recruitment and activation of the serine/threonine kinase, ROCK, which leads to downstream activation of myosin light chain, increased F-actin formation, and contraction.Early studies showed that antigen-induced AHR, an exaggerated and excessive narrowing of the airways in response to stimuli, resulted in elevated RhoA levels in mice and rats^4,5.Importantly, treatment with a RhoA inhibitor, Clostridium botulinum C3 exoenzyme, blocked acetylcholine-induced AHR. A recent study found that the RhoGEF, Arhgef12, was highly expressed in patients with asthma, treatment of tracheal rings with RhoGEF inhibitors showed decreased contractility, and in an asthma model involving house dust mite allergic sensitization, Arhgef12-KO mice had significantly decreased AHR⁶. This study supports the role of RhoA signaling in AHR and identifies novel targets for potential intervention.

RhoA signaling has also been shown to affect other features of asthma, such as airway remodeling^{(reviewed in 3)}. This signaling pathway contributes to enhanced airway narrowing in asthma through a multitude of ways^{(reviewed in 3)}, but critically, through enhanced Ca2+-sensitization-induced contraction of ASMCs. Several signaling molecules have been shown to regulate RhoA-mediated ASMC contraction; for example, work by the Sakai lab recently identified the lncRNA, MALAT1, as a positive regulator of RhoA protein expression in ASMCs⁷. Mechanistically, they found that MALAT1 regulates RhoA by inhibiting miR-133a-3p in ASMCs to augment hypercontractility. Cytokine-driven activation of RhoA has also been shown to drive remodeling in asthma, and a recent study linked cytokines IL-13 and IL-17A to β1 integrin activation, which led to enhanced force transmission in ASMCs in a RhoA-dependent fashion⁸. Other force transmission and cytoskeletal fluidization mechanisms also contribute to ASMC contraction in asthma^{(reviewed in 9)}. In addition to augmenting ASMC contraction, RhoA also regulates airway remodeling phenotypes such as ASMC proliferation and migration in response to an array of stimulants, such as VEGF¹⁰ and kisspeptin¹¹.

RhoA role in airway inflammation in asthma

Airway inflammation involves the interplay among immune cells, epithelial cells, and ASMCs via various cytokines and mediators¹²; for instance, work by Ramos-Barbón et al. described how CD4+ T cells drive ASMC remodeling in experimental asthma¹³ (see figure 1). Another study also found significant crosstalk between CD4+ T cells and ASMCs, but in a pediatric obesity-related asthma model¹⁴. In addition to CD4+ T cells, eosinophils, mast cells, and other immune cells are augmented in asthmatic patients¹⁵. There is a body of work suggesting that the RhoA/ROCK signaling pathway can influence immune cell function in a multitude of disease settings^{(reviewed in 16)}. One recent example identified a paradoxical function for neutrophils, where they release anti-inflammatory extracellular vesicles, which are formed by RhoA-dependent budding¹⁷. Importantly, that was not in the setting of airway inflammation, but there are several specific examples where the RhoA/ROCK signaling does regulate immune cells to promote airway inflammation³.

For example, ROCK-depleted mice challenged with ovalbumin to produce an inflammatory response similar to asthma, displayed decreased IL-13 and IL-5 cytokine levels, lower eosinophil counts, and reduced AHR compared to wild-type mice¹⁸. In another study, RhoA levels were shown to be elevated in eosinophils of children with allergic asthma, and RhoA inactivation attenuated allergen-induced eosinophil infiltration into allergic airway-inflamed mouse lungs¹⁹.Work by the Zhang lab sought to deduce how obesity increases the severity of AHR in asthma, and identified the G-protein coupled receptor 40 (GPCR40) as a critical protein that responded to long-chain fatty acids to exacerbate several features of asthma²⁰.Furthermore, inhibition of GPR40 with a small-molecule antagonist, DC260126, reduced TH2 cytokine levels, decreased inflammatory cell infiltration, and reduced AHR; all of these measurements also correlated with a downregulation in RhoA and ROCK in their model²⁰.Collectively, these studies suggest that RhoA/ROCK signaling in immune cells is also important for controlling airway inflammation in asthma.

RhoA as a potential asthma therapeutic

While corticosteroids provide relief of chronic airway inflammation, they do not reverse all pathological effects associated with asthma.Targeting the RhoA/ROCK pathway in pre-clinical studies showed tremendous promise towards reversing acetylcholine or ovalbumin-AHR, and while the ROCK inhibitor Y-27632 was not as effective as a β2‐adrenoreceptor agonist for acute asthma treatment, it may be a viable additive therapy as it works by a completely different mechanism²¹. Several other ROCK inhibitor studies have shown positive results in attenuating risk factors of asthma, like airway mucus hypersecretion and inflammation, through the modulation of key cytokines^22,23. A recent study corroborated these findings in another allergic asthma animal model, and found that the ROCK inhibitor hydroxyfasudil reduced the concentration of Th2 cytokine IL-13 while reducing inflammatory cell levels to provide improved airway remodeling²⁴. In 2007, lovastatin was shown to reduce hyperresponsiveness in a rat model of asthma, and the benefit seen by lovastatin appeared to be RhoA-dependent²⁵. Recently, the transgelin-2 targeting molecule, TSG1180, was shown to have beneficial effects for treating asthma, and in part occurred through altering the phosphorylation states of ROCK and RhoA²⁶. One last example identified that the vitamin E isoform, β-tocotrienol molecule, was capable of inhibiting PDGF-BB-induced proliferation and migration of ASMCs, which it achieved through the inhibition of RhoA activation and ROS production²⁷.

Summary and Future Insights

RhoA appears to play an integral role in several key pathologies associated with asthma and undoubtedly is a critical regulator of ASMCs and immune cells in the setting of airway inflammation, airway narrowing, and AHR. Remarkably, new data suggest that RhoA may even affect other cells involved in asthma, such as mesenchymal stem cells²⁸. Several promising studies using ROCK inhibitors have shown a critical role for RhoA/ROCK signaling in asthma progression in pre-clinical studies, and it will be fascinating to see if viable RhoA/ROCK-targeting therapeutics eventually emerge for the treatment of asthma.

References

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